Regulation of protein synthesis at the level of mRNA translation is important for: (1) viral shut-off of host protein synthesis, (2) rapid adjustments to changing red blood cell maturation. Translational regulation involves changes in the phosphorylation state of the initiation factor elF-2. We have therefore studied the role of elF-2 phosphorylation during protein synthesis initiation, and its regulation by the adenylate energy change, the pyridine nucleotide redox state and hemin, in reticulocyte lysate and intact cells. We have identified and characterized four distinct components which regulate the steady state phosphorylation level and activity of the initiator methionyl-tRNAf binding protein eIF-2, (1) eIF-2 alpha, (2) eIF-2 phosphatase, (3) conformational state eIF-2 and (4) binding of eIF-2 to other translational components. Determination of the eIF-2 pool size, direct chemical measurement of the eIF-2 phosphorylation state, and correlation of these with extent and kinetics of onset of translational inhibition now show that while phosphorylation of eIF-2 does not directly inhibit its activity, phosphorylation is required for additional modification of eIF-2 which results in conversion of catalytic to stoichiometric utilization of eIF-2.